The human plasma membrane protein, P-glycoprotein (Pgp) is an ATP-driven drug-exporting pump that counteracts chemotherapy in cancer cells and limits the bioavailability of therapeutic drugs in other tissues. Modulation of the drug-exporting activity of Pgp could improve cancer treatments and AIDS treatment. The overall goal of this project is to elucidate the molecular mechanism of drug transport by Pgp. Utilizing new techniques for studying drug transport and detailed structural analysis, a molecular model of the transport mechanism has been generated. In this proposal, this model will be tested and refined using mutagenesis of human Pgp and quantitative kinetic, thermodynamic, spectroscopic and computational methods of enzyme analysis. The model postulates that the coupling of ATP hydrolysis to drug transport involves concerted conformational movements initiated at the nucleotide sites and transmitted through the intracellular domains that lead to transmembrane helix rotations, and rearrangements that drive the drug through the protein channel(s). In Specific Aim 1, drug channel(s) will be functionally characterized in thermodynamic and transport studies to distinguish between alternative models for the reaction cycle of Pgp. Novel spin-labeled transport substrates will be synthesized to facilitate these studies. In Specific Aim 2, the hydration exchange model of drug transport, which associates drug binding and coupled transport events with conformational changes will be refined. Drug-binding sites will be mapped to the structure of P-glycoprotein using mutagenesis and assessment of functional consequences and physical properties of the drug-binding sites will be characterized. In Specific Aim 3 dynamic structure/function relationships will be located by observing conformational changes associated with coupled drug transport events through the application of EPR spectroscopic techniques. Conformational changes during the reaction cycle will be determined for 1) residues involved in initial drug binding at the membrane interface and 2) transmembrane-helix 6 (TM6) involved in drug release at the other side of the membrane. Knowledge acquired in these studies will be used to achieve a rigorous, dynamic, molecular description of coupled drug transport by P-gp that could aid in the rational design of drugs and methodologies to overcome or modulate this transporter. [unreadable] [unreadable]